ABSTRACT
【Objective】 To study the molecular mechanism of 9 samples with rare RhD variants and their RhD epitopes and protein structure. 【Methods】 The 9 blood samples with rare RhD variants were collected from 210 644 blood donors of Shenzhen Blood Center. Regular serological assaying was used for determination of Rh type for the 9 samples. Indirect anti-human globulin test (IAT) was used to confirm the RhD antigen and to screen the antibodies. D-screen reagent was sued to analyze the RhD epitopes of the samples. RHD zygosity testing of the samples was detected by PCR-SSP. The nucleotide sequences of all 9 exons and adjacent flanking intron regions of RHD gene were sequenced. The prediction of the effects of mutations on RhD protein function were analyzed using PROVEAN, SIFT, PolyPhen-2 and MutationTaster software. Robetta and Swiss-PdbViewer 4.1.0 were used for modeling the tertiary structures of RhD. 【Results】 A total of 9 individuals with rare RhD variants were identified as follows: RHD*weak D type 25, RHD*weak D type 50, RHD*weak D type 95, RHD*weak D type 12, RHD*weak D type 128 and four novel RHD alleles. The prediction of the tertiary structures showed that the RhD protein conformation was disrupted in the 9 rare RhD variants samples. 【Conclusion】 Five rare and four novel RHD alleles have been identified. Their phenotypic and genotypic descriptions enrich the database of reported RHD alleles. Bioinformatics analysis provided evidences for further study of the structure and functions of RhD protein.
ABSTRACT
【Objective】 To explore the characteristics of the D antigen epitope of individuals with RhD variants and the genetic molecular mechanism of gene mutations in Guangzhou. 【Methods】 A total of 59 samples of RhD variants were collected from blood donors and hospitals in Guangzhou from January to August 2019. Serological characteristics of D epitopes were further analyzed using two kinds of monoclonal anti-D reagents and D epitope detection kits, and RHCE phenotypic typing was performed. QuickGene DNA extraction kit was used to extract the genomic DNA of the samples, and PCR-RFLP method was used to analyze the RHD gene zygote type. The RHD gene sequence was detected by multiple ligation-dependent probe amplification(MLPA) genotyping, and the RHD exon(1~10) Sanger sequencing was performed on the samples still in doubt after the above detection. DNAStar/SeqMan analysis software was used for comprehensive analysis. 【Results】 In this group of individuals with RhD variants in Guangzhou, 27.12%(16/59) were detected from blood donors [accounting for 0.007%(16/232 793) of blood donors in Guangzhou during the same period], and difficult samples of patients sent by hospitals for determination accounted for 72.88%(43/59). RHD genotype detection: 40.68%(24/59) were RHD*weak partial 15, 25.42%(15/59) were RHD* DⅥ.3 and 33.90%(20/59) were rare RHD variants [76.92%(10/13) were RhD variants with 2 different alleles]. Serological D-screen revealed a relatively fixed pattern of RHD*DⅥ.3 in anti-D antibody(clone: P3*212 23B10), while the others was negative. The phenotypic distribution of RhD variant CE was Ccee 38.98%(23/59), ccEe 35.59%(21/59), CcEe 25.42%(15/59). 【Conclusion】 Weak partial D15 and DⅥ.3 were the most common RhD variants in Guangzhou Han population, and DⅥ can be preliminarily identified by serological methods such as D-Screen anti-D reagent, while the remaining RhD variants can only be identified by molecular biological methods, and >95% of the RhD variants were C+ or E+ phenotypes.